A. Accumulation of 1As3PGA in everted membrane vesicles prepared from E. coli strain AW3110 harboring either plasmid pArsJ (●,○) or vector plasmid pUC19 (▼) was assayed as described in Experimental procedures using 1 mg/ml of membrane protein. To produce 1AS3PGA in situ, the reaction mixture contained, 5 units of commercial rabbit GAPDH, 10 mM DTT, 5 mM NAD+ and 5 mM As(V). The reaction was initiated by addition of 5 mM G3P, final concentration. 10 μM FCCP, final concentration, was added at the indicated time (arrow).
B. The requirements for in situ generation of 1As3PGA were determined by addition of the components of the GAPDH reaction. (Δ), GAPDH, NAD+ and G3P; (●), GAPDH and G3P; (○), GAPDH and NAD+; (▼), NAD+ and G3P. Vesicular arsenic was determined by ICP-MS. Data are the mean±SE (n=3).